Most therapeutic compounds are delivered to the target organ or tissue through the circulation. However, in most cases the drug or other treatment will not only target the diseased organ or tissues, but will also be taken up by other organs and tissues in the body. This can result in undesirable side effects due to, for example, generalized toxic effects throughout the patient's body. Thus, it would be desirable to selectively target specific organs or tissues. In addition, coupling of a therapeutic compound to a targeting molecule can improve the uptake properties of the compound into the targeted tissue or cells, resulting in a more effective molecule. Therefore, coupling to targeting molecules yields compounds that are more effective and less toxic than the parental compound, see Curnis et al., 2000, Nature Biotechnol. 18, 1185-1190. This can be applied to a wide range of compounds, such as peptides, proteins, cytostatic agents, antibiotic and antiviral agents.
In the case of muscle diseases such as Duchenne muscular dystrophy (DMD), myotonic dystrophy (MD) or spinal muscular atrophy (SMA), muscle-specific peptides can be conjugated to, for example, antisense oligonucleotides (AONs) and small interfering RNA (siRNA). AONs and siRNAs have high potency to be applied as new classes of medicines for treatment of specific diseases by blocking undesired gene transcription. In the field of DMD therapy antisense-induced exon skipping is gaining attention as a novel and promising tool for correction of the translational reading frame of the dystrophin transcript. The aim is to manipulate splicing in such a manner that the targeted exon will be skipped (through binding of the AONs to pre-mRNA) and a slightly shorter but in-frame transcript will be generated. This would allow correction of the translational reading frame, and induction of the synthesis of a Becker muscular dystrophy (BMD)-like dystrophin protein that may significantly alleviate progression of the disease.
Several reports have shown the therapeutic potential of the exon skipping strategy for restoring dystrophin production in cultured patient-derived muscle cells in vitro (van Deutekom et al., 2001, Hum. Mol. Genet. 10, 1547-1554) and in transgenic hDMD mouse muscle tissue in vivo by intramuscular injections (Bremmer-Bout et al., 2004, Mol. Ther. 10, 232-240). However, the biggest hurdle to overcome is the poor in vivo muscle uptake of these AONs, especially in all kind of myopathies like Myotonic Dystrophy (MD) and Spinal Muscular Atrophy (SMA).
An efficient therapy for these muscle wasting diseases will require that essentially all of the skeletal muscles including those of arms and legs and the muscles involved in respiration as well as the cardiac muscle are targeted. None of the mechanisms investigated to date have the ability to specifically deliver (antisense) oligonucleotides, let alone entire genes, to essentially all muscle tissues/cells simultaneously over the entire body. Methods for the in vivo delivery of genes or other compounds into muscle that have been published so far include injection of naked DNA with or without electrotransfer, use of microbubbles (Lu et al. 2003, Gene Ther. 10, 396-405) and systemic delivery using poloxamer (a hydroxypoly(oxy-ethylene)poly(oxypropylene)). Recently it was shown in mdx mice that systemic delivery of morpholino AONs resulted in an increased dystrophin expression in several muscles (Alter et al., 2006, Nature Med. 12, 1-3). However, even after repeated administration, dystrophin expression was barely detectable in diaphragm and was undetectable in heart muscle. Furthermore, in these mdx mice the AONs are taken up rather easy into the muscles because the muscle membranes are compromised, which is not the case for the muscles of, for instance, young Duchenne patients. Also, in other muscle diseases like SMA and MD delivery of AON is complicated due to the fact that in this case the muscle cell walls are not compromised.
Ideally, whole-body muscle therapy would use systemic delivery (e.g. intravenously or subcutaneously) of a compound endowed with a cell specific targeting ability. Some molecules have been described that have potential for muscle cell targeting. The first report is of a peptide sequence with enhanced in vivo skeletal and cardiac muscle binding, that was identified by screening a random phage display library (Samoylova and Smith, 1999, Muscle Nerve 22, 460-466). However, it has not yet been shown whether or not this peptide can be used for in vivo targeting of conjugated compounds to muscle cells. Also a number of 7-mer peptide sequences that were recovered from human skeletal muscle after in vivo screening of phage random peptide library have been described (Arap et al., 2002, Nature Medicine 8, 121-127). No information is given on binding to cardiac muscle cells. Also here it has not yet been shown whether or not these peptides can be used for in vivo targeting of conjugated compounds to muscle cells. Another molecule that has been described is an Fv part of a monoclonal antibody (mAb) that is selectively transported into skeletal muscle in vivo (Weisbart et al., 2003, Mol. Immunol. 39, 783-789). Single chain Fv fragments of the murine mAb were injected into the tail veins of mice and 4 hours later the fragments were found in 20% of skeletal muscle cells, primarily localized in the nucleus. It was shown that the mAb binds to the protein myosin IIb in lysates of skeletal muscle cells, but it did not bind any protein in lysates of heart muscle cells. Therefore, this antibody might be useful for targeting to skeletal muscles, but not to the heart muscle.
In the case of lysosomal storage disease a problem in the enzyme replacement therapy is poor in vivo uptake of the therapeutic recombinant enzyme into the muscle cells. For example in Pompe's disease (glycogen storage disease type II) the doses of recombinant human acid α-glucosidase (rhGAA) that were needed in clinical studies were very high, due to poor uptake of the rhGAA into the skeletal muscle (Winkel et al., 2004, Ann. Neurol. 55, 495-502). In light of the above, it is very clear that further improvements in delivery systems are necessary to achieve specific uptake of agents such as AONs in vivo.